Abstract By optically sensing the mid-infrared absorption induced photothermal effect, midinfrared photothermal (MIP) microscope enables super-resolution IR imaging and scrutinizing of biological systems in an aqueous environment. However, the speed of current lock-in based sample-scanning MIP system is limited to 1.0 millisecond or longer per pixel, which is insufficient for capturing dynamics inside living systems. Here, we report a single pulse laserscanning MIP microscope that dramatically increases the imaging speed by three orders of magnitude. We harness a lock-in free demodulation scheme which uses high-speed digitization to resolve single IR pulse induced contrast at nanosecond time scale. To realize single pulse photothermal detection at each pixel, we employ two sets of galvo mirrors for synchronized scanning of mid-infrared and probe beams to achieve an imaging line rate over 2 kHz. With video-rate imaging capability, we observed two types of distinct dynamics of lipids in living cells. Furthermore, by hyperspectral imaging, we chemically dissected a single cell wall at nanometer scale. Finally, with a uniform field of view over 200 by 200 μm 2 and 2 Hz frame rate, we mapped fat storage in free-moving C. elegans and live embryos.